Tibor Krenacs

Tissue Healing by Regeneration

and Repair with Fibrosis

Medical Students 2015/16

- Neutralization

of toxic agents

- Elimination of

toxic agents

+ tissue debrie

INFLAMMATION REGENERATION

- Restoration of tissue

structure and function

(Damage: caused by

trauma and/or toxic

agents + inflammation)

FIBROSIS

-Tissue repair

when reticular fibers &

basement membranes

(ECM) damaged

(Cause: Serious injury,

chronic inflammation)

INJURY HEALING

Tissue integrity – essential for life

Regeneration

Inflammation: serious injury; innate + adaptive immunity, proteases, fibrosis

Fibrotic scarringe.g. myocardial infarction

„ischemic hearth disease”huge burden public health

7.4 million death/2012

Amphibians e.g. salamandra regenerate extremities

Blastema cells – undifferentiated stock of stem cells (proximal neurons + Schwann cells)

Embryonal wound healing – without scarring

Brock J…Martin P (UCL): J Cell Biol 1996 135:1097-1107

Day-18th chicken embryo – wing bud incision wound Similar in human embryo before the 3rd trimester

• TGF3 > TGF-1-2; Reverses after birth

• Immature vs mature immune system

Regeneration – Repair with Fibrosis

- cascades of overlapping events

- interactions between cell-cell and cell-matrix

- Bleeding - Coagulation: Isolation from the environment

- Inflammation: Prevention of infection/septicemia

- Debridement: Elimination of tissue debrie

- Migration, proliferation: Replenishment of lost tissue

Skin wound as a model

- Epithelialization: Parenchyma regeneration

- Angiogenesis: Nutrition of granulation tissue

- Fibroplasia: Fibroblast invasion, matrix production

- Remodelling: Generation and degeneration of ECM

- Contraction: Close up wound edges

- Resolution: Restoration of appearance and function

Repair of specific tissues

Pathology of tissue repair

HEALING: Aiming to regenerate tissue structure and function

REGENERATION REPAIR with FIBROSIS

Cell proliferation – Cell cycle

Promoters

-Cyclin-cdk

complexes

Inhibitors

-Cyclin dependent

kinase inhibitors

APOPTOSIS

programmed

cell death

Growth factors

Licensing

E2F

RetinoblastomaRb phosphorylation

DNA REPAIR

2015

Nobel price

in chemistry

T. Lindahl

P. Modrich

A. Sancar

Comitted

normal cell

population

BALANCE

1. symmetric

2. assymetric

Regulation of baseline cell population

proliferationapoptosis

cell death

• assymetric cell division

• self-renewal

Cells able to regenerate throughout life

STEM CELLS

INJURY!

elevated

cell decay

Intermitotic

cells

reversibly

postmitotic

cells

STEM CELLS

Embryonic stem cell

Pluripotent: inner cell mass cells of morula (ecto-, meso-, entoderm)

Maintain the compartment

regeneration

Form different tissues

In vitro fertilization

MHC - histocompatibility

Transplant rejection

Adult stem cells

Multipotent: some cell types e.g. bone marrow (or mesenchymal) stem cells

can form all subtypes of hemopoietic cells

Unipotent: skin, GI track eptithelial stem cells

Reprograming - Induced pluripotent stem cells (iPS)

2012 Nobel prize in Physiology-Medicine:

Sir John B. Gurdon and Shinya Yamanaka

"for the discovery that mature cells can be

reprogrammed to become pluripotent"

J. Embryol. Exp. Morphol. 10, 622-640. 1962

Reprograming

Oct3/4, Sox-2

c-Myc, Klf4, Nanog

Therapeutic

cloning

CD34+

stem

cells

REGENERATIVE MEDICINE

Regeneration - proliferative capacity of parenchymal cells

labile cells/tissues: continuously replicating – Intermitotic cells

>1.5% of the cells in mitosis, may be destroyed by sublethal injury

- hemopoiesis, skin, oral mucosa, GI tract, respiratory, urinary tract

Stem cells – multipotent/unipotent – Efficient regeneration

Ki67

Stem cell „niche” (microenvironment)

Cornea regeneration after excimer laser ablation

6 h

12 h 24 h

Laser

Dr. Imola Ratkay-Traub MD PhD: FDA approval of femtosecond laser (LASIK)

(Prof. Tibor Juhasz, Univ. Michigan, Ann Arbor)

Cornea regeneration 24h post laser treatment

EGFR – growth factor receptorProliferation – Ki67 protein

Control – Resting cornea

Limbal – under regeneration

Connexin direct cell-communication

compartmental functions – cell network

Stroma

Epithel

Permanent cells/tissues: terminally differentiated cells, no proliferative capacity

(postmitotic cells) - neurons of the central nervous system, myocardium

- if injured repaired by connective tissue scar

(skeletal muscle? – satellite cells)

Stabil cells/tissues: terminally differentiated G0 phase cells

(reversibly postmitotic) - adapt well, high metabolic activity, longevity

- liver, kidney, pancreas, endometrium, endocrine glands

- endothelial cells, fibroblasts, smooth muscle

No stem cells (except in liver), differentiated cells can proliferate

Prometheus’s punishment by Zeus – eagle eating from his liverLiver regeneration

Myocardium (permanent cells): complex function, longevity, no regeneration

no stem cells, the same for endocardium; Healing with scar: - arrhytmias

- damaged pumping function

Healing of special tissues

Acut myocardial necrosisMyocardial fibrosis post-infartion

fibrosis

Nervous systhem: a neurons can not replicate (SVZ, hyppocampus dentate gyrus)

Central Peripheral

- No axon regeneration - Axon regeneration if broken ends

- Repair involves microglia, astrocyte aligned accurately; if not traumatic

proliferation (inflammation) neuroma: fibrosis, abortive axon prolif.

Permanent? cell/tissue, BUT

satellite cells (stem cells)

If matrix (endo-, perimysium) preserved

muscle fibers can fully regenerate

(however, long cells, stroma is broken - fibrosis)

S

Satellite

cell (S)

Skeletal muscle regeneration

myoblastTRAUMA

Skeletal muscle regeneration

*

24h

Mp

Mp

E

Rat m.soleus

Notexin

(venom of the

tiger snake)

Day-7 Central nucleus

Zoltan Takacs

Growth factor receptor subtypes

Protein kinase

(growth factors)

G-protein coupled receptor

(kemokines)Without intrinsic

kinase activity

(cytokines)

2012 Nobel Prize in Chemistry

Robert J. Lefkowitz and Brian K. Kobilka

"for studies of G-protein-coupled receptors"

Sun et al. Nature. 2011,

480(7377): 372–375.

Blocking of inhibitory factors

of retinal axon regeneration

Regeneration medicine

PTEN – Akt/mTOR SOCS3 – JAK/STAT3

Growth factors in wound healing

EGF

inhibitor

Fibroblast

proliferation

migration

ECM synthesis

Keratinocyte

proliferation

differentiation

Angiogenesis

Epithelial proliferation EGF, TGF-α, KGF, HGF

Monocyte chemotaxis PDGF, FGF, TGF-β

Fibroblast migration PDGF, FGF, TGF-β

Fibroblast proliferation PDGF, EGF, FGF, TNF

Angiogenesis VEGF, Ang, FGF-2

Collagen synthesis TGF-β, PDGF

(TGF-β3 inhibits)

EGFR

Skin

BM stroma (myelofibrosis)

(proliferation, migration, ECM production, kemotaxis, vasodilatation)

Growth factors in wound healing

Chemokines, cytokines are also involved in the inflammatory phase of regeneration,

which are not discussed here, only their receptor types were shown.

Extracellular matrix (ECM) and cell-matrix interactions

Functions:

Sequesters water and provides turgor (resistance) for tissues

minerals (Ca10(PO4)6(OH)2 hidroxil-apatit, for bone rigidity

Supports (and anchores) parenchymal cells and their migration

Basement membrane (BM) in kidney is involved in glomerular filtration

Binds and offers regulatory molecules (growth factors)

for: cell proliferation, migration, differentiation

Initiates signal transduction through integrins

They can be transmembrane molecules too (syndecan, collagen XVII)

Active contributors of wound healing

Types: Interstitial matrix

Basement membrane

ECM is a dynamic continuously remodelled complex of macromolecules

ECM not an inert material just for filling up gaps !!!

Extracellular matrix (ECM)

1. Fibrillar proteins: collagens, elastin (elastase – Ilona Banga)

2. Water-hydrated gels: Proteoglycans, hyaluronan

3. Multiadhesive proteins: fibronectin, laminin

Signals from the matrix through integrins: laminin, fibronectin, collagens

Heparan sulfate proteoglycans: RTK receptor coreceptor

Basal membrane

Laminin

Cornea

Skin

Collagen I and III fibers

Proteoglycans

Laminin

fibers

Laminin

fibers

Growth factors Growth factors

Growth factor

receptor

Growth factor

receptor

Collagen Collagen

FibronectinFibronectin

ECM signaling

through integrins

FOC

ECM binds and offers growth factors for cell activation

Timing of main events in wound healing

Trichrome staining

Collagen - blueGranulation tissue

Early Late

- Early fibroblasts & ECM

- Network of newly

formed vessels

- Inflamatory cells

(macrophages)

Re-epithelialization

Epidermal keratinocytes produced from interfollicular (basal) and follicular

(bulge region) stem cells migrate from both edges of wound

Keratinocytes migrate over the transitional wound matrix and fibrin cloth

(including early matrix components e.g. fibronectin)

Covering the wound surface below scab, keratinocytes differentiate into stratified

squamous „neoepidermis” which usually show hyperplasia

Re-epithelialization is phylogenetically a robust feature under most conditions

Delayed re-epitelialization

-epithelial hyperplasia

(EGF, TGF-α, KGF)C-Met (HGFR) mutantControl – „Normal” wounding

Formation of new vessels

- Vasodilataion, endothelial cells budding (sprouting), proliferation

- Progresses toward wound space (hypoxia gradient & growth factors),

release of pericyctes

- Immature vessels differentiate into capillaries, arterioles,

and venules (regain pericyte support)

Upon angiogenic stimuli e.g. by macrophages (VEGF, FGF) and keratinocytes (VEGF)

PDGF, Angiogenin (maturation)

Angiogenesis

Fibroblasts (PDGF, FGF-2, TGF-β1 –β2, TGF–β3)

- Migrate into wound site and replicate (stabile cells)

- Dominant cell type at wound edge

- Synthesize and deposit collagen and proteoglycans

• Matrix deposition depends on oxygen and substrate availability and

regulated by growth factors

• Cross linking of collagen is important for tensile strength, which requires vitamin C

• Vitamin C-defficiency: skeletal deformities, hemorrages and retarded wound healing

Fibroplasia – Matrix production

Loose connective tissue

Mesenhcymal cell

Elastic fibers: fibrillin scaffold + elastin

Marfan syndrome: fibrillin mutations

- long thin body, long extremities

- highly flexible joints (shoulder, elbow, ankle)

- extra long wingspan: (>1.05 of height)

Aorta dissections: detachment of the aorta layers

Epithelial migration - Matrix remodelling

Coll XVII

E-Cadherin

angiogenezis

inhibitors

Collagen XVIII - endostatin

Perlecan - endorepellin

Cleavage of precursors results in new biological activity

(controlled proteolyzis – bioactive molecular fragments)

Martricryptic sites

Collagenases, Gelatinases, Strome-lyzinesMMPs (Zn2+):

MMP: Matrix metalloproteinases

TIMP: Tissue inhibitor of MMPs

TIMPs

Dedifferentiation Redifferentiation

Fibrin clot

PROLIFERATION - MIGRATION

BM - synthesis

Keratinocyte

differentiationEPIDERMIS

Basal

membrane

DERMIS

Integrin

Coll XVII

Coll VII

BM-Laminin

Coll I, III

MMP1,2,3, 9,10,11

Matrix cleavage

Plasmin (MMP activation)

EGFR

TGF-

HGFLM-1

LM-5Plasmin LM-5

LM-332

Neutrophil

granulocytes

Clotting

Fibrous union

Cell proliferation

Granulation tissue

Macrophages

Fibroblasts

New capillaries

SCAB

Contraction

Resolution

Types of healing

Identical steps, but

in the secondary:

- defect is larger

- wound edge is wider

- extensive inflammation

- healing is slower

- scar is larger

- substantial contraction

Dependent from: - type of tissue

- regeneration potential

- level of ECM damage

Liver: - regeneration (focal, zonal necrosis)

- damage of ECM network (large abscess,

cirrhosis) healing by fibrosis

Healing of special tissues

CK7+ Hering ducts

(oval cells are stem cells)

Hepatocytes + oval cells

The pathway of healing is identical with that in the skin but faster

Skin transplanted into the oral cavity - wound - healed with scar

Oral mucosa – Healing without scar (fibrosis)

Larjava H: J Can Dent Assoc 2011;77:b18

Day-3 Day-7 Day-60

Differences:

(inherent) - less inflammatory cells (granulocytes, marcrophages)

- saliva: antibacterial protection, accelerated clotting (exosomes)

& proliferation (EGF), leukocyte protease inhibitor, histatins

- less protocollagen I and fibronectin, more tenascin

- residens fibroblasts express less TGF1 and decorin (fibrosis)

- less fibrillogenesis and myofibroblasts (contraction)

TraumaBleeding, coagulation (fibrin)

Inflammation, Fibroblasts

PDGF

Bone and cartillage

progenitor cells (chondrocytes & osteoblasts)

From periosteum & bone marrow

Synthesis of new matrix

Soft callus (cartillage)

Endochondrial ossification

(similar to that of epiphyseal

growth plate - calcification)

Woven bone – lamellar bone

-trabecular bone: Remodelling

Regeneration of bone fracture

Factors influencing wound healing

Local: type of tissue, blood supply (nutrients, oxigenization), mechanical stress,

localization & type of injury (cut, large destruction, acid, alkaline, burn)

irradiation: UV, radioactive (ionizing) etc…

Systhemic: cardiovascular status, infections, nutrition (malnutrition),

diabetes, lack of vitamins (e.g. vitamin C), corticosteroids

Complications

Large tissue loss, deep wound – substantial scarring and distortion

Pressure ulcer (decubitus): insufficient perfusion and oxigenization

mechanical pressure > resistance of arterial wall

Insufficient closure, dehiscence: mechanical stress/trauma, increased

abdominal pressure due to coughing or vomiting; Risk: diabetes, obesity, poor suture

Old age, poor perfusion: poor oxygenization, mechanical stress, diabetes

Chronic wound (diabetic ulcer): arteriopathia, reduced perfusion,

oxygen and nutrients,insufficient immune response against infection, neuropathy

Enzymes of the larvae digest dead tissue at wound edges

which is then eaten up by the larvae

Support the elimination even of antibiotic resistant bacteria:

Staphylococcus aureus (MRSA) or Streptococcus B

„Debridement”

„Maggot” therapy: Larvae of Lucilia sericata fly into open wound

Elimination of dead tissue which support microbial growth

Used from ancient times:

Egyipt, Roman Empire

Aboriginals in Australia

During World Wars

Studied at J Hopkins U.1929

Keloid: elevated collagen synthesis, lost control during healing

inherited, more frequent in the african-american populaltion

Contracture of palmar fascias: (Dupuytren contrature) restricted

mobility/extendibility of fingers due to elevated fibrosis and shortening of fascia around

finger tendons (post-trauma)

(alkali drinking - suicide)

Colliquation necrosis

Oesophagus stricture

10% NaOH kontroll

Osman et al. 2008

INJURY

COAGULATION

PLATELETS

INFLAMMATION DEBRIDEMENT

PREVENTION of

INFECTION

MACROPHAGES

GRANULOCYTESFIBROBLASTS

„REMODELLING”

COLLAGEN

LYSISCOLLAGEN

SYNTHESIS

EPITHELIUMANGIOGENESIS

Myofibroblasts

PROTEOGLYCAN

SYNTHESIS

HEALED WOUND (Resolution)

Myofibroblasts Contraction

Regeneration-Healing

Coordinated interactions between cells,

ECM, growth factors, enzymes

(proteases, protein kinases).

Thank you!

Lung fibrosis – Trichrome staining (blue = collagen)